CN104447787A - Method for separating and purifying two sesquiterpene lactone compounds from ambrosia artemisiifolia - Google Patents
Method for separating and purifying two sesquiterpene lactone compounds from ambrosia artemisiifolia Download PDFInfo
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- CN104447787A CN104447787A CN201410853228.5A CN201410853228A CN104447787A CN 104447787 A CN104447787 A CN 104447787A CN 201410853228 A CN201410853228 A CN 201410853228A CN 104447787 A CN104447787 A CN 104447787A
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- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/10—Spiro-condensed systems
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/04—Ortho-condensed systems
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Abstract
A method for separating and purifying two sesquiterpene lactone compounds from ambrosia artemisiifolia L includes the steps that alcohol extraction is carried out, an ethyl acetate extraction layer removes pigments through porous adsorption resin after petroleum ether and ethyl acetate extraction, after separation achieved by means of ODS C-18 reversed phase column chromatography and sephadex gel filtration chromatography, recrystallization is carried out through petroleum ether and acetone, and high-purity mono compounds of psilostachyin and psilostachyin B are obtained. The method is easy to operate, the porous adsorption resin, ODS C-18 and sephadex gel which are used can be used repeatedly, and it is verified through the experiment that the two obtained compounds have good killing effect on the invasive specie of pomacea canaliculata lamarck, can be used for preventing the pomacea canaliculata lamarck and have good development prospects.
Description
Technical field
The invention belongs to cultural control field, specifically, is the methods and applications of a kind of separation and purification two kinds of sesquiterpene lactones compound psilostachyin and psilostachyin B from artemisiifolia.
Background technology
Fushou spiral shell (Pomacea canaliculata Lamarck) originates from South America, the Changjiang river areas to the south is mainly distributed in China, mainly taking food rice tillering tender shoots and waterplant, is a kind of invasion hydrocoles very serious to agriculture production harm.The current control to Fushou spiral shell is mainly based on Agro-chemicals control, and as niclosamide and metaldehyde etc., but chemical agent is high except cost, outside comparatively large to other non-target organism toxicity, also to ecological environmental pollution seriously, affects agriculture safety in production.Artemisiifolia (Ambrosia artemisiifoliaL.) is the malignant weed of invading China in recent years, can have a strong impact on agriculture production, and biolytic diversity.Therefore, utilizing this pernicious instruction plant of artemisiifolia to kill Fushou spiral shell, effectively can control Fushou spiral shell, can contain spreading of artemisiifolia to a certain extent again, is " combatting poison with poison ", " double gain ".
Existing patent CN103004892A reports that artemisiifolia dry powder has toxic action to Fushou spiral shell, and the artemisiifolia dry powder adding 10g/L in water body can kill the Fushou spiral shell of 95% after 3 days, shows artemisiifolia and has and kill Fushou spiral shell application prospect preferably.But this patent does not relate in artemisiifolia the activeconstituents killing Fushou spiral shell, so far, research report also be there is no to the activeconstituents killing Fushou spiral shell in artemisiifolia both at home and abroad.Based on this, the present invention carries out separation and purification to the activeconstituents of artemisiifolia further, provide a kind of from this plant rapid extraction be purified into the method for poisoning Fushou spiral shell activeconstituents.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, provides a kind of method of the two kinds of sesquiterpene lactones compounds of separation and purification from artemisiifolia easy and simple to handle, provides the application of these two kinds of compounds in control Fushou spiral shell simultaneously.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of method of separation and purification two kinds of sesquiterpene lactones compounds from artemisiifolia, and the method step is as follows:
A, get artemisiifolia over-ground part, clean, air-dry, be ground into granularity≤20 object powder, the ratio of the ethanol of 3-7ml 95% concentration expressed in percentage by volume is added in 1g artemisiifolia powder, ethanol at room temperature lixiviate 3 times is added in artemisiifolia powder, each lixiviate 48-96 hour, filters, merging filtrate, concentrating under reduced pressure obtains paste alcohol steep thing after reclaiming ethanol;
B, add the ratio of 30-60ml water in 1g alcohol steep thing, alcohol steep thing is added water-dispersion, 3 times are extracted respectively in normal temperature successively again by isopyknic sherwood oil and ethyl acetate, each extraction time is 3-5 hour, obtain petroleum ether extraction layer and extraction into ethyl acetate layer, reclaim under reduced pressure sherwood oil and acetic acid ethyl acetate extract, obtain sherwood oil medicinal extract and ethyl acetate extract; Wherein, sherwood oil and ethyl acetate are all analytical pure;
C, be adsorbed to saturated through little macroporous adsorbent resin after saturated solution by ethyl acetate extract dissolve with methanol, first wash with water to after colourless, then use the methanol-eluted fractions of 90% concentration expressed in percentage by volume to colourless, collect elutriant, concentratedly to obtain enriched material;
As long as above-mentioned little macroporous adsorbent resin can be used for removing ethyl acetate cream pigment all can, as MCI GELCHP 20P 37-75um or SMB MCI GEL 50-70um etc.
D, ODS C-18 reverse-phase chromatographic column on enriched material is carried out chromatography, be that the methyl alcohol of 3:7 → 7:3 and the mixed solution of water carry out gradient elution by volume ratio, the elutriant when volume ratio of collection methyl alcohol and water is 4:6, detect through thin-layer chromatography, merge same principle point cut, obtain two components, this two component is gone up respectively sephadex column (Sephadex LH-20) column chromatography and carry out purifying, wash-out is carried out with methyl alcohol, detect through thin-layer chromatography, merge principal point cut and concentrating under reduced pressure, obtain two solid state things, be sherwood oil and the acetone recrystallization of 1:1 mixing more respectively by volume ratio by gained solid, obtain monomeric compound 1 and 2,
E, employing proton nmr spectra and carbon-13 nmr spectra technology carry out the comparison of Structural Identification and physico-chemical property to above-mentioned monomeric compound 1 and 2, and confirmation gained monomeric compound 1 is psilostachyin, and monomeric compound 2 is psilostachyin B.
Empirical tests, above-mentioned two monomeric compound psilostachyin and psilostachyin B have toxic action to Fushou spiral shell, and therefore, the present invention provides the application of above-mentioned two monomeric compounds in control Fushou spiral shell simultaneously.
So, the present invention obtains the main active ingredient that two are killed Fushou spiral shell from artemisiifolia, makes to develop activeconstituents Billy in artemisiifolia more efficient, controlled with artemisiifolia dry powder control Fushou spiral shell; Present method is easy and simple to handle, and the little macroporous adsorbent resin of filler, ODS C-18, dextrane gel etc. that use can be reused, and cost is low, pollutes little.Further, artemisiifolia belongs to vegetable material, utilize its extract for preventing and treating Fushou spiral shell be a kind of with evil control harmful ecological preventions, effectively can not only prevent and treat Fushou spiral shell, and spreading of artemisiifolia can be controlled; Recycling for harmful instruction plant opens new approach, and research that is ecological for invasion and biotic intrusion control techniques has great reference value.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1
1. the extraction and isolation of compound
Get the air-dry artemisiifolia over-ground part being ground into powder of 10kg, with the alcohol steep 3 times of 50L 95% concentration expressed in percentage by volume under room temperature, each lixiviate 50 hours, filter, merging filtrate, after filtrate reduced in volume reclaims ethanol, obtain paste alcohol steep thing 500g, this 500g alcohol steep thing is added 20L water-dispersion, use isopyknic sherwood oil successively, extraction into ethyl acetate, each extraction 3 times, each extraction 4 hours, obtain petroleum ether extraction layer and extraction into ethyl acetate layer, reclaim under reduced pressure sherwood oil and acetic acid ethyl acetate extract, obtain sherwood oil medicinal extract 90g, ethyl acetate extract 90g, be saturated solution by ethyl acetate extract dissolve with methanol, be adsorbed to saturated through little macroporous adsorbent resin (MCI GEL CHP20P 37-75um), first wash with water to after colourless, then use the methanol-eluted fractions of 90% concentration expressed in percentage by volume to colourless, collect elutriant, concentrated, gained enriched material is crossed ODS C-18 reverse-phase chromatography column chromatography, with methanol/water (3:7 → 7:3, v/v) gradient elution is carried out, each concentration collects 10 bottles, every bottle of 100mL, collect methyl alcohol: water=4:6 (v/v) elution fraction, detect through thin-layer chromatography, merge same principle point cut and namely merge 1-3 bottle and 5-9 bottle, obtain two components, this two component is utilized respectively Sephadex LH-20 column chromatographic isolation and purification (carrying out wash-out with methyl alcohol), detect through thin-layer chromatography, merge principal point cut and concentrating under reduced pressure, obtain two solid state things, they are utilized sherwood oil/acetone (1:1 respectively further, v/v) recrystallization, obtain monomeric compound 1 (60mg) and 2 (90mg) of two colorless needle crystals, detecting purity through HPLC is more than 95%.
2. the Structural Identification of compound
Above-claimed cpd 1, be colourless acicular crystal, nuclear magnetic resonance data is as follows:
1h NMR (500MHz, CDCl
3) δ 6.27 (H-11a, d, J=3.2Hz, 1H), 5.58 (H-11b, d, J=3.2Hz, 1H), 4.99 (H-8, d, J=9.5Hz, 1H), 3.43 (C-H, m, 1H), 2.90-1.49 (m, complex signal), 1.23 (H-5, s, 3H), 1.06 (H-1, d, J=7.5Hz, 3H).
13c NMR (126MHz, CDCl
3) δ 177.32 (C4), 169.68 (C12), 139.00 (C11), 121.77 (C13), 93.64 (C5), 83.42 (C8), 79.58 (C1), 41.72 (C7), 40.17 (C10), 30.21 (C6), 27.30 (C3), 26.94 (C9), 15.04 (C2), 14.85 (C14), 14.64 (C15). contrast document, its
1hNMR and
13cNMR data are consistent with psilostachyin, and crystalline melting point 213 °, optical value in physico-chemical property
(c 0.5, CHCl
3) consistent with bibliographical information (CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1993,18 (3): 164-166), so be accredited as same thing further, namely deterministic compound 1 is psilostachyin.
Above-claimed cpd 2 is colourless acicular crystal,
1hNMR composes display: there are 2 typical unimodal methyl signals [δ
h1.54 (s, H
3-14) and 1.44 (s, H
3-15)], there is 1 typical lactone bond [δ in C-6
h4.88 (d, J=9.1Hz, H-6)], C-11 exists α, and the outer subunit group of ring of the unsaturated gamma lactone form of β ', has 1 to the strong bimodal proton [δ that influences each other
h6.25 (d, J=3.5Hz, H-13a) and 5.48 (d, J=3.1Hz, H-13b)].
1h NMR (400MHz, Pyr) δ 6.25 (1H, d, J=3.5Hz, H-13a), 5.48 (1H, d, J=3.1Hz, H-13b), 4.88 (1H, d, J=9.1Hz, H-6), 3.30 (1H, m, H-7), 2.68 (1H, ddd, J=16.9, 9.3, 6.2Hz, H-9a), 2.50 (1H, ddd, J=16.9, 6.9, 5.5Hz, H-9b), 2.42 – 2.28 (2H, m, H-2), 2.24 – 2.16 (2H, m, H-3), 1.87 (1H, ddd, J=14.2, 9.5, 4.8Hz, H-8a), 1.67 – 1.58 (1H, m, H-8b), 1.54 (3H, s, H-14), 1.44 (3H, s, H-15).
13c NMR (100MHz, Pyr) δ 171.27 (C-4), 170.64 (C-12), 139.83 (C-11), 133.61 (C-1), 126.71 (C-10), 120.57 (C-13), 87.42 (C-5), 84.19 (C-6), 42.09 (C-7), 35.18 (C-3), 30.51 (C-8), 26.30 (C-9), 25.80 (C-2), 23.87 (C-14), 23.21 (C-15). contrast document, its
1hNMR and
13cNMR data are consistent with psilostachyin B, are accredited as same thing, and crystalline melting point 118 °, optical value in physico-chemical property
(c 0.7, CHCl
3) consistent with bibliographical information (Phytochemistry, 1986,25 (6): 1355-1358), so be accredited as same thing further, namely deterministic compound 2 is psilostachyin B.
The structural formula of above-mentioned two compound psilostachyin and psilostachyin B is:
Embodiment 2 two monomeric compounds of the present invention are tested the poisoning of Fushou spiral shell
Fushou spiral shell poisoning experiment adopts infusion method.Two compounds the present invention prepared are mixed with the aqueous solution of 5 groups of different concns respectively, are placed in Glass Containers; Often organize input 25 healthy Fushou spiral shells, complete submergence, prevents Fushou spiral shell from escaping at Glass Containers lid last layer gauze.In time processing 24h, 48h, 72h, take out the individuality of doubtful death respectively, with distilled water recovery 30min, what open in 30min that operculum creeps is spiral shell of living, and spies unresponsively to be decided to be dead individuals.Arrange blank, each process repeats 3 times simultaneously, and experimental temperature is 26 DEG C.The results are shown in following table 1.
Table 1 artemisiifolia sesquiterpene lactones is to the cytotoxicity of just incubating Fushou spiral shell
Above-mentioned experimental result shows, under the concentration of psilostachyin 25mg/L, after 3 days, the mortality ratio of Fushou spiral shell reaches 100%; Under the concentration of psilostachyin B 25mg/L, after 3 days, the mortality ratio of Fushou spiral shell also reaches 88.9%, illustrate that psilostachyin and psilostachyin B has obvious cytotoxicity to Fushou spiral shell, both compare, psilostachyin better effects if.
Claims (5)
1. the method for separation and purification two kinds of sesquiterpene lactones compounds from artemisiifolia, it is characterized in that, the method step is as follows:
A, get artemisiifolia over-ground part, clean, air-dry, be ground into powder, the ratio of the ethanol of 3-7ml 95% concentration expressed in percentage by volume is added in 1g artemisiifolia powder, ethanol at room temperature lixiviate 3 times is added in artemisiifolia powder, each lixiviate 48-96 hour, filters, merging filtrate, concentrating under reduced pressure obtains paste alcohol steep thing after reclaiming ethanol;
B, add the ratio of 30-60ml water in 1g alcohol steep thing, alcohol steep thing is added water-dispersion, 3 times are extracted respectively in normal temperature successively again by isopyknic sherwood oil and ethyl acetate, each extraction time is 3-5 hour, obtain petroleum ether extraction layer and extraction into ethyl acetate layer, reclaim under reduced pressure sherwood oil and acetic acid ethyl acetate extract, obtain sherwood oil medicinal extract and ethyl acetate extract;
C, be adsorbed to saturated through little macroporous adsorbent resin after saturated solution by ethyl acetate extract dissolve with methanol, first wash with water to after colourless, then use the methanol-eluted fractions of 90% concentration expressed in percentage by volume to colourless, collect elutriant, concentratedly to obtain enriched material;
D, ODS C-18 reverse-phase chromatographic column on enriched material is carried out chromatography, gradient elution is carried out with the mixed solution that methyl alcohol and water are 3:7 → 7:3 by volume, the elutriant when volume ratio of collection methyl alcohol and water is 4:6, detect through thin-layer chromatography, merge same principle point cut, obtain two components, this two component is gone up SephadexLH-20 column chromatography respectively and carries out purifying, wash-out is carried out with methyl alcohol, detect through thin-layer chromatography, merge principal point cut and concentrating under reduced pressure, obtain two solid state things, be sherwood oil and the acetone recrystallization of 1:1 mixing more respectively by volume ratio by gained solid, obtain monomeric compound 1 and 2,
E, employing proton nmr spectra and carbon-13 nmr spectra technology carry out the comparison of Structural Identification and physico-chemical property to above-mentioned monomeric compound 1 and 2, and confirmation gained monomeric compound 1 is psilostachyin, and monomeric compound 2 is psilostachyin B.
2. the method for separation and purification two kinds of sesquiterpene lactones compounds from artemisiifolia as claimed in claim 1, it is characterized in that, the little macroporous adsorbent resin in described step C is MCI GEL CHP20P 37-75um or SMB MCIGEL 50-70um.
3. the method for separation and purification two kinds of sesquiterpene lactones compounds from artemisiifolia as claimed in claim 1, is characterized in that, artemisiifolia Powder Particle Size≤20 order in described steps A.
4. the application of the monomeric compound psilostachyin as described in claim 1-3 in control Fushou spiral shell.
5. the application of the monomeric compound psilostachyin B as described in claim 1-3 in control Fushou spiral shell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017044860A1 (en) * | 2015-09-11 | 2017-03-16 | El-Sawy Mohamed Fakhr El-Din | Medicinal ambrosia plant extracts |
| CN114671837A (en) * | 2022-04-11 | 2022-06-28 | 沈阳农业大学 | Eucalyptus alkane type sesquiterpenoids in ragweed and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1882247A (en) * | 2003-11-13 | 2006-12-20 | 康普顿发展有限公司 | Molluscicidal and anti-barnacle compounds |
| CN103004892A (en) * | 2012-12-10 | 2013-04-03 | 华南农业大学 | Application of alien invasive plant Ambrosia artemisiifolia in control of golden apple snail |
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2014
- 2014-12-31 CN CN201410853228.5A patent/CN104447787B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1882247A (en) * | 2003-11-13 | 2006-12-20 | 康普顿发展有限公司 | Molluscicidal and anti-barnacle compounds |
| CN103004892A (en) * | 2012-12-10 | 2013-04-03 | 华南农业大学 | Application of alien invasive plant Ambrosia artemisiifolia in control of golden apple snail |
Non-Patent Citations (1)
| Title |
|---|
| 王浦海 等: "豚草化学成分的研究", 《中国药学杂志》, vol. 18, no. 3, 31 March 1993 (1993-03-31), pages 164 - 166 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017044860A1 (en) * | 2015-09-11 | 2017-03-16 | El-Sawy Mohamed Fakhr El-Din | Medicinal ambrosia plant extracts |
| CN114671837A (en) * | 2022-04-11 | 2022-06-28 | 沈阳农业大学 | Eucalyptus alkane type sesquiterpenoids in ragweed and preparation method and application thereof |
| CN114671837B (en) * | 2022-04-11 | 2023-04-14 | 沈阳农业大学 | Eucalyptus alkane type sesquiterpene compound in ragweed and preparation method and application thereof |
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